- Email: [email protected]
Orai1 and TRPC3 in platelets from patients with type 2 diabetes mellitus
Blood Cells, Molecules, and Diseases 43 (2009) 211–213
Contents lists available at ScienceDirect
Blood Cells, Molecules, and Diseases j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y b c m d
Enhanced expression of STIM1/Orai1 and TRPC3 in platelets from patients with type 2 diabetes mellitus Hanene Zbidi a, José J. López b, Nidhal Ben Amor a, Aghleb Bartegi a, Ginés M. Salido b, Juan A. Rosado b,⁎ a b
Institute of Biotechnology, University of Monastir, Monastir, Tunisia Department of Physiology, University of Extremadura, Av. Universidad s/n. Cáceres 10071, Spain
a r t i c l e
i n f o
Article history: Submitted 11 March 2009 Available online 17 May 2009 (Communicated by M. Lichtman, M.D., 14 April 2009) Keywords: Platelets Type 2 diabetes mellitus Transient receptor potential canonical 3 STIM1 Orai1
a b s t r a c t Type 2 diabetes mellitus (DM2) is a metabolic syndrome that contributes to both macrovascular and microvascular disorders, where platelet hyperaggregability, associated to abnormal intracellular Ca2+ homeostasis, plays an important role. We have now investigated the expression of different proteins associated to Ca2+ entry, a major Ca2+ signalling event. DM2 donors were randomly selected from normotensive patients with glycosylated Hb levels (HbA1c) over 6%. Control subjects were normal age- and gender-matched healthy people with HbA1c levels in the normal range (3.5–5%). Expression of TRPC1, 3 and 6, STIM1 and Orai1 was analyzed by Western blotting in DM2 patients and controls. Expression of TRPC1 in platelets from DM2 donors and controls was similar; however, expression of TRPC6 is reduced in platelets from DM2 patients as compared to healthy controls. We have found that expression of TRPC3, Orai1 and STIM1 is enhanced in DM2 subjects as compared to controls. Our findings provide an explanation to the enhanced Ca2+ entry induced by physiological agonists in platelets from DM2 patients. © 2009 Elsevier Inc. All rights reserved.
Introduction Type 2 diabetes mellitus (DM2) is a metabolic chronic disease that leads to several cardiovascular complications, where platelet hyperaggregability and hyperactivity have been shown to play an important role [1]. Among the intracellular pathways associated with platelet dysfunction in DM2 patients, Ca2+ mobilisation, specially Ca2+ entry, which is a crucial event in platelet physiology [2,3], has been reported to be altered [4,5]. In non-excitable cells, such as platelets, Ca2+ entry can be achieved by different mechanisms. Capacitative Ca2+ entry (CCE), a mechanism regulated by the Ca2+ content of the intracellular stores, is a major mechanism for Ca2+ influx in platelets, a process that has been proposed to occur with the participation of the intraluminal Ca2+ sensor, STIM1, the membrane protein Orai1 and certain members of the TRPC subfamily of channels, such as TRPC1 and TRPC6 [6–8]. There are also a number of reports supporting the role of TRPC6 in noncapacitative Ca2+ entry (NCCE) mechanisms, as well as TRPC3 [9]. Ca2+ entry in platelets stimulated by the physiological agonist thrombin or by passive depletion of the intracellular stores using the SERCA inhibitor thapsigargin in DM2 subjects has been shown to be enhanced [10], which might be involved in platelet hyperactivity; however, the precise underlying mechanism remains unclear. In the present study we have investigated the expression of STIM1, Orai1 and ⁎ Corresponding author. Fax: +34 927 257110. E-mail address: [email protected] (J.A. Rosado). 1079-9796/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bcmd.2009.04.005
the TRPC proteins 1, 3 and 6, involved in Ca2+ entry in DM2 patients, and we have compared the level of expression of these proteins with those in platelets from healthy subjects. Materials and methods Materials Apyrase (grade VII), aspirin, anti-actin antibody and bovine serum albumin were from Sigma (Madrid, Spain). Anti-TRPC1 antibody was from Alomone (Jerusalem, Israel). Anti-TRPC3 antibody was from Abcam (Cambridge, U.S.A.). Anti-TRPC6 antibody and horseradish peroxidase-conjugated goat anti-rabbit IgG antibody were from Santa Cruz (Santa Cruz, CA, U.S.A.). Anti-STIM1 was from BD Transduction Laboratories (NJ, U.S.A.). Anti-Orai1 was purchased from ProSci Inc. (CA, U.S.A.). Enhanced chemiluminescence detection reagents were from Pierce (Cheshire, U. K.). Hyperfilm ECL and horseradish peroxidase-conjugated ovine anti-mouse IgG antibody were from Amersham (Arlington Heights, IL, U.S.A.). All other reagents were purchased from Panreac (Barcelona, Spain). Subjects Patients with type 2 diabetes mellitus and not showing other pathologies and healthy drug-free volunteers were randomly obtained from normotensive patients of the Clinical Analysis Laboratory, Cáceres, Spain. Informed consent was obtained from
212
H. Zbidi et al. / Blood Cells, Molecules, and Diseases 43 (2009) 211–213
every subject. Blood was obtained at 9:00 AM, in accordance with the Declaration of Helsinki, from 15 healthy and 15 DM2 subjects. Blood glucose concentration in DM2 patients was in the range of 180 to 240 mg/dL. Glycosylated Hb levels (HbA1c) of DM2 patients, used as an index of metabolic control, was N6%. Control subjects were normal age- and gender-matched healthy people that had HbA1c levels in the normal range (3.5–5%). Platelet preparation Platelets were prepared as described previously [10]. Briefly, blood was drawn by venepuncture from drug-free volunteers and mixed with one-sixth volume of acid/citrate dextrose anticoagulant containing (in mM): 85 sodium citrate, 78 citric acid and 111 D-glucose. Platelet-rich plasma was then prepared by centrifugation for 5 min at 700 ×g and aspirin (100 μM) and apyrase (40 μg/mL) added. Cells were then collected by centrifugation at 350 ×g for 20 min and resuspended in HEPES-buffered saline containing (HBS in mM): 145 NaCl, 10 HEPES, 10 D-glucose, 5 KCl, 1 MgSO4, pH 7.45 and supplemented with 0.1% w/v bovine serum albumin and 40 μg/mL apyrase.
top panel; p b 0.05; n = 8). Similar results were obtained when Orai1 expression was investigated in platelets from DM2 and healthy subjects (Orai1 expression in DM2 was 122 ± 14% of controls; Fig. 1B, top panel; p b 0.05; n = 8). Orai1 and STIM1 have been reported as major components of CCE in platelets [8]. The increase in the expression of both proteins concomitantly might explain the enhanced CCE induced by store depletion using thapsigargin [10]. Despite the amount of STIM1 and Orai1 was found to be enhanced in platelets from DM2 patients, detection of TRPC1 was found to be slightly reduced in platelets from DM2 as compared to age- and gender-matched healthy donors (TRPC1 expression in DM2 was 92 ± 5% of controls; Fig. 1C, top panel; n = 8). A more significant attenuation in the level of expression in platelets from DM2 patients was found for TRPC6, whose expression in DM2 was 66 ± 7% of the expression in controls; Fig. 1D, top panel; p b 0.05; n = 6). In human platelets, both TRPC1 and TRPC6 have been shown to participate in the conduction of CCE [7,14]. Endogenously expressing TRPC1 interacts with Orai1 and STIM1 in a complex where Orai1 mediates the communication between STIM1 and hTRPC1, which is essential for the activation of hTRPC1 subunits in the CCE mode [7]. TRPC6 has been shown to be involved both in CCE and NCCE pathways in platelets and
Western blotting Western blotting was performed as previously described [11]. Cell samples (2 × 108 cells/mL) from DM2 or healthy donors were mixed with an equal volume of 2 × Laemmli's buffer [12] with 10% dithiothreitol followed by heating for 5 min at 95 °C. Proteins were separated by 10% SDS-PAGE (50 μg total protein loaded/sample) and electrophoretically transferred, for 2 h at 0.8 mA/cm2, onto nitrocellulose for subsequent probing. Blots were incubated overnight with 10% (w/v) BSA in Tris-buffered saline with 0.1% Tween 20 (TBST) and membranes were then incubated with the anti-TRPC1, anti-TRPC3 or anti-TRPC6 antibody diluted 1:200 in TBST for 2 h, the anti-STIM1 antibody diluted 1:250 in TBST for 2 h or the anti-Orai1 antibody diluted 1:1000 in TBST for 90 min. To detect the primary antibody, blots were incubated with the appropriate horseradish peroxidaseconjugated anti-rabbit IgG or anti-mouse IgG antibody, diluted 1:10,000 in TBST, and exposed to enhanced chemiluminescence reagents for 4 min. Blots were then exposed to photographic films and the optical density was estimated using scanning densitometry. For reprobing, membranes were incubated for 30 min at 50 °C in stripping buffer containing 100 mM 2-mercaptoethanol, 65.5 mM Tris, and 2% SDS, pH 6.7. Membranes were then washed and Western blotting was performed using anti-actin antibody diluted 1:1000 for 2 h as described previously. Statistical analysis Data are expressed as mean ± SEM. Significant difference between individual groups was tested by using the Student's t-test. The level of significance was set at p b 0.05. Results and discussion We have previously reported that Ca2+ entry in platelets is enhanced in DM2 patients compared to controls [10,13]. Here, we have investigated and compared in platelets from DM2 and healthy subjects the expression of different membrane proteins involved in CCE and NCCE mechanisms for Ca2+ influx, including the endoplasmic reticulum Ca2+ sensor, STIM1, and the Ca2+ permeable channel subunits Orai1 and TRPC1, as well as that of TRPC6, which has been shown to participate in CCE as well as NCCE in human platelets, and the TRPC3 protein involved in NCCE [7], by Western blotting. A STIM1immunoreactive band of about 87 kDa was detected in platelet lysates. Interestingly, the amount of STIM1 detected in platelets from DM2 patients was found to be enhanced to 131 ± 12% of controls (Fig. 1A,
Fig. 1. Expression of STIM1, Orai1 and TRPC1 and 6 in platelets from DM2 patients and control subjects. Platelet lysates from control and DM2 donors were subjected to SDS/ 10%-PAGE and Western blotting with anti-STIM1 (A, top panel), anti-Orai1 (B, top panel), anti-TRPC1 (C, top panel) and anti-TRPC6 (D, top panel) antibodies. Membranes were reprobed with anti-actin antibody (bottom panels) for protein loading control. Histograms represent protein expression as percentage of healthy controls and are expressed as average ± SEM of six to eight separate experiments. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel.
H. Zbidi et al. / Blood Cells, Molecules, and Diseases 43 (2009) 211–213
213
agonists, and might overcome the reduced expression of TRPC6, which might explain the altered ionic homeostasis described in insulin resistant conditions [19]. Acknowledgments This work was supported by MCyT-DGI grant BFU2007-60104, Fundesalud (Junta de Extremadura) grant PRI08A003 and Ministerio de Asuntos Exteriores y Cooperación (A/016208/08). J.J.L. was supported by a grant (BFU2004-00165) from the Spanish MEC-DGI. References
Fig. 2. Expression of TRPC3 in platelets from DM2 patients and control subjects. Platelet lysates from control and DM2 donors were subjected to SDS/10%-PAGE and Western blotting with anti-TRPC3 antibody (top panel). Membranes were reprobed with antiactin antibody (bottom panel) for protein loading control. Histograms represent protein expression as percentage of healthy controls and are expressed as average ± SEM of six separate experiments. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel.
other cells [7,15]. We have recently found that the interaction of Orai1/ STIM1 with TRPC6 recruits TRPC6 in the CCE pathway [16]. To test protein loading in samples from DM2 and healthy control donors we performed Western blotting with an anti-actin antibody. As shown in Fig. 1, bottom panels, Western blotting with anti-actin antibody confirmed a similar amount of actin in both lanes (DM2 and controls), which, together with the equal cell concentration used for both experimental groups (2 × 108 cells/mL) support that the different protein detection observed in DM2 and healthy donors by Western blotting is unlikely attributed to different protein loading but to a modification induced by DM2 itself. We have further investigated the expression of other NCCE channel subunits, such as TRPC3, also expressed in human platelets [7]. Here we show that TRPC3 expression was significantly enhanced in platelets from DM2 patients compared to controls (TRPC3 expression in platelets from DM2 patients was 125 ± 8% of that in platelets from healthy donors; Fig. 2, top panel; p b 0.05; n = 6). Western blotting of the same membranes with anti-actin antibody confirms a comparable protein loading in both lanes (Fig. 2, bottom panel). We report for the first time altered expression of a number of proteins involved in CCE and NCCE in platelets from DM2 patients compared with age- and gender-matched healthy donors. Our findings provide an explanation to the altered Ca2+ entry in response to the physiological agonist thrombin or thapsigargin previously reported in platelets from DM2 patients [10]. Thrombin and thapsigargin are able to induce both CCE and NCCE [7,17], the latter (thapsigargin) by stimulating secretion of platelet-activating factors [18]. The increased expression of the CCE complex STIM1/Orai1 and that of the NCCE channel TRPC3 might explain the enhanced Ca2+ entry observed in platelets from DM2 subjects upon stimulation with
[1] M.E. Carr, Diabetes mellitus: a hypercoagulable state, J. Diabetes Complicat. 15 (2001) 44–54. [2] T.J. Rink, S.O. Sage, Calcium signaling in human platelets, Annu. Rev. Physiol. 52 (1990) 431–449. [3] I. Jardin, N. Ben Amor, J.M. Hernandez-Cruz, G.M. Salido, J.A. Rosado, Involvement of SNARE proteins in thrombin-induced platelet aggregation: evidence for the relevance of Ca2+ entry, Arch. Biochem. Biophys. 465 (2007) 16–25. [4] V. Randriamboavonjy, F. Pistrosch, B. Bolck, R.H. Schwinger, M. Dixit, K. Badenhoop, R.A. Cohen, R. Busse, I. Fleming, Platelet sarcoplasmic endoplasmic reticulum Ca2+-ATPase and mu-calpain activity are altered in type 2 diabetes mellitus and restored by rosiglitazone, Circulation 117 (2008) 52–60. [5] J.A. Rosado, F.R. Saavedra, P.C. Redondo, J.M. Hernandez-Cruz, G.M. Salido, J.A. Pariente, Reduced plasma membrane Ca2+-ATPase function in platelets from patients with non-insulin-dependent diabetes mellitus, Haematologica 89 (2004) 1142–1144. [6] I. Jardin, J.J. Lopez, G.M. Salido, J.A. Rosado, Orai1 mediates the interaction between STIM1 and hTRPC1 and regulates the mode of activation of hTRPC1-forming Ca2+ channels, J. Biol. Chem. 283 (2008) 25296–25304. [7] I. Jardin, P.C. Redondo, G.M. Salido, J.A. Rosado, Phosphatidylinositol 4,5bisphosphate enhances store-operated calcium entry through hTRPC6 channel in human platelets, Biochim. Biophys. Acta 1783 (2008) 84–97. [8] A. Braun, D. Varga-Szabo, C. Kleinschnitz, I. Pleines, M. Bender, M. Austinat, M. Bosl, G. Stoll, B. Nieswandt, Orai1 (CRACM1) is the platelet SOC channel and essential for pathological thrombus formation, Blood 113 (2009) 2056–2063. [9] G.M. Salido, S.O. Sage, J.A. Rosado, TRPC channels and store-operated Ca2+ entry, Biochim. Biophys. Acta 1793 (2009) 223–230. [10] F.R. Saavedra, P.C. Redondo, J.M. Hernandez-Cruz, G.M. Salido, J.A. Pariente, J.A. Rosado, Store-operated Ca2+ entry and tyrosine kinase pp60(src) hyperactivity are modulated by hyperglycemia in platelets from patients with non insulindependent diabetes mellitus, Arch. Biochem. Biophys. 432 (2004) 261–268. [11] P.C. Redondo, M.T. Harper, J.A. Rosado, S.O. Sage, A role for cofilin in the activation of store-operated calcium entry by de novo conformational coupling in human platelets, Blood 107 (2006) 973–979. [12] P.C. Redondo, G.M. Salido, J.A. Pariente, J.A. Rosado, Dual effect of hydrogen peroxide on store-mediated calcium entry in human platelets, Biochem. Pharmacol. 67 (2004) 1065–1076. [13] A. Bouaziz, S. Salido, P.J. Linares-Palomino, A. Sanchez, J. Altarejos, A. Bartegi, G.M. Salido, J.A. Rosado, Cinnamtannin B-1 from bay wood reduces abnormal intracellular Ca2+ homeostasis and platelet hyperaggregability in type 2 diabetes mellitus patients, Arch. Biochem. Biophys. 457 (2007) 235–242. [14] G.M. Salido, S.O. Sage, J.A. Rosado, Biochemical and functional properties of the store-operated Ca2+ channels, Cell. Signal. 21 (2009) 457–461. [15] T. Hofmann, A.G. Obukhov, M. Schaefer, C. Harteneck, T. Gudermann, G. Schultz, Direct activation of human TRPC6 and TRPC3 channels by diacylglycerol, Nature 397 (1999) 259–263. [16] I. Jardin, L.J. Gomez, G.M. Salido, J.A. Rosado, Dynamic interaction of hTRPC6 with the Orai1/STIM1 complex or hTRPC3 mediates its role in capacitative or noncapacitative Ca2+ entry pathways, Biochem J (2009) doi:10.1042/BJ20082179. [17] J.A. Rosado, S.O. Sage, Protein kinase C activates non-capacitative calcium entry in human platelets, J. Physiol. 529 (Pt. 1) (2000) 159–169. [18] A.G. Harper, S.L. Brownlow, S.O. Sage, A role for TRPV1 in agonist-evoked activation of human platelets, J. Thromb. Haemost. 7 (2009) 330–338. [19] L.M. Resnick, Ionic basis of hypertension, insulin resistance, vascular disease, and related disorders. The mechanism of “syndrome X”, Am. J. Hypertens. 6 (1993) 123S–134S.
Contents lists available at ScienceDirect
Blood Cells, Molecules, and Diseases j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y b c m d
Enhanced expression of STIM1/Orai1 and TRPC3 in platelets from patients with type 2 diabetes mellitus Hanene Zbidi a, José J. López b, Nidhal Ben Amor a, Aghleb Bartegi a, Ginés M. Salido b, Juan A. Rosado b,⁎ a b
Institute of Biotechnology, University of Monastir, Monastir, Tunisia Department of Physiology, University of Extremadura, Av. Universidad s/n. Cáceres 10071, Spain
a r t i c l e
i n f o
Article history: Submitted 11 March 2009 Available online 17 May 2009 (Communicated by M. Lichtman, M.D., 14 April 2009) Keywords: Platelets Type 2 diabetes mellitus Transient receptor potential canonical 3 STIM1 Orai1
a b s t r a c t Type 2 diabetes mellitus (DM2) is a metabolic syndrome that contributes to both macrovascular and microvascular disorders, where platelet hyperaggregability, associated to abnormal intracellular Ca2+ homeostasis, plays an important role. We have now investigated the expression of different proteins associated to Ca2+ entry, a major Ca2+ signalling event. DM2 donors were randomly selected from normotensive patients with glycosylated Hb levels (HbA1c) over 6%. Control subjects were normal age- and gender-matched healthy people with HbA1c levels in the normal range (3.5–5%). Expression of TRPC1, 3 and 6, STIM1 and Orai1 was analyzed by Western blotting in DM2 patients and controls. Expression of TRPC1 in platelets from DM2 donors and controls was similar; however, expression of TRPC6 is reduced in platelets from DM2 patients as compared to healthy controls. We have found that expression of TRPC3, Orai1 and STIM1 is enhanced in DM2 subjects as compared to controls. Our findings provide an explanation to the enhanced Ca2+ entry induced by physiological agonists in platelets from DM2 patients. © 2009 Elsevier Inc. All rights reserved.
Introduction Type 2 diabetes mellitus (DM2) is a metabolic chronic disease that leads to several cardiovascular complications, where platelet hyperaggregability and hyperactivity have been shown to play an important role [1]. Among the intracellular pathways associated with platelet dysfunction in DM2 patients, Ca2+ mobilisation, specially Ca2+ entry, which is a crucial event in platelet physiology [2,3], has been reported to be altered [4,5]. In non-excitable cells, such as platelets, Ca2+ entry can be achieved by different mechanisms. Capacitative Ca2+ entry (CCE), a mechanism regulated by the Ca2+ content of the intracellular stores, is a major mechanism for Ca2+ influx in platelets, a process that has been proposed to occur with the participation of the intraluminal Ca2+ sensor, STIM1, the membrane protein Orai1 and certain members of the TRPC subfamily of channels, such as TRPC1 and TRPC6 [6–8]. There are also a number of reports supporting the role of TRPC6 in noncapacitative Ca2+ entry (NCCE) mechanisms, as well as TRPC3 [9]. Ca2+ entry in platelets stimulated by the physiological agonist thrombin or by passive depletion of the intracellular stores using the SERCA inhibitor thapsigargin in DM2 subjects has been shown to be enhanced [10], which might be involved in platelet hyperactivity; however, the precise underlying mechanism remains unclear. In the present study we have investigated the expression of STIM1, Orai1 and ⁎ Corresponding author. Fax: +34 927 257110. E-mail address: [email protected] (J.A. Rosado). 1079-9796/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bcmd.2009.04.005
the TRPC proteins 1, 3 and 6, involved in Ca2+ entry in DM2 patients, and we have compared the level of expression of these proteins with those in platelets from healthy subjects. Materials and methods Materials Apyrase (grade VII), aspirin, anti-actin antibody and bovine serum albumin were from Sigma (Madrid, Spain). Anti-TRPC1 antibody was from Alomone (Jerusalem, Israel). Anti-TRPC3 antibody was from Abcam (Cambridge, U.S.A.). Anti-TRPC6 antibody and horseradish peroxidase-conjugated goat anti-rabbit IgG antibody were from Santa Cruz (Santa Cruz, CA, U.S.A.). Anti-STIM1 was from BD Transduction Laboratories (NJ, U.S.A.). Anti-Orai1 was purchased from ProSci Inc. (CA, U.S.A.). Enhanced chemiluminescence detection reagents were from Pierce (Cheshire, U. K.). Hyperfilm ECL and horseradish peroxidase-conjugated ovine anti-mouse IgG antibody were from Amersham (Arlington Heights, IL, U.S.A.). All other reagents were purchased from Panreac (Barcelona, Spain). Subjects Patients with type 2 diabetes mellitus and not showing other pathologies and healthy drug-free volunteers were randomly obtained from normotensive patients of the Clinical Analysis Laboratory, Cáceres, Spain. Informed consent was obtained from
212
H. Zbidi et al. / Blood Cells, Molecules, and Diseases 43 (2009) 211–213
every subject. Blood was obtained at 9:00 AM, in accordance with the Declaration of Helsinki, from 15 healthy and 15 DM2 subjects. Blood glucose concentration in DM2 patients was in the range of 180 to 240 mg/dL. Glycosylated Hb levels (HbA1c) of DM2 patients, used as an index of metabolic control, was N6%. Control subjects were normal age- and gender-matched healthy people that had HbA1c levels in the normal range (3.5–5%). Platelet preparation Platelets were prepared as described previously [10]. Briefly, blood was drawn by venepuncture from drug-free volunteers and mixed with one-sixth volume of acid/citrate dextrose anticoagulant containing (in mM): 85 sodium citrate, 78 citric acid and 111 D-glucose. Platelet-rich plasma was then prepared by centrifugation for 5 min at 700 ×g and aspirin (100 μM) and apyrase (40 μg/mL) added. Cells were then collected by centrifugation at 350 ×g for 20 min and resuspended in HEPES-buffered saline containing (HBS in mM): 145 NaCl, 10 HEPES, 10 D-glucose, 5 KCl, 1 MgSO4, pH 7.45 and supplemented with 0.1% w/v bovine serum albumin and 40 μg/mL apyrase.
top panel; p b 0.05; n = 8). Similar results were obtained when Orai1 expression was investigated in platelets from DM2 and healthy subjects (Orai1 expression in DM2 was 122 ± 14% of controls; Fig. 1B, top panel; p b 0.05; n = 8). Orai1 and STIM1 have been reported as major components of CCE in platelets [8]. The increase in the expression of both proteins concomitantly might explain the enhanced CCE induced by store depletion using thapsigargin [10]. Despite the amount of STIM1 and Orai1 was found to be enhanced in platelets from DM2 patients, detection of TRPC1 was found to be slightly reduced in platelets from DM2 as compared to age- and gender-matched healthy donors (TRPC1 expression in DM2 was 92 ± 5% of controls; Fig. 1C, top panel; n = 8). A more significant attenuation in the level of expression in platelets from DM2 patients was found for TRPC6, whose expression in DM2 was 66 ± 7% of the expression in controls; Fig. 1D, top panel; p b 0.05; n = 6). In human platelets, both TRPC1 and TRPC6 have been shown to participate in the conduction of CCE [7,14]. Endogenously expressing TRPC1 interacts with Orai1 and STIM1 in a complex where Orai1 mediates the communication between STIM1 and hTRPC1, which is essential for the activation of hTRPC1 subunits in the CCE mode [7]. TRPC6 has been shown to be involved both in CCE and NCCE pathways in platelets and
Western blotting Western blotting was performed as previously described [11]. Cell samples (2 × 108 cells/mL) from DM2 or healthy donors were mixed with an equal volume of 2 × Laemmli's buffer [12] with 10% dithiothreitol followed by heating for 5 min at 95 °C. Proteins were separated by 10% SDS-PAGE (50 μg total protein loaded/sample) and electrophoretically transferred, for 2 h at 0.8 mA/cm2, onto nitrocellulose for subsequent probing. Blots were incubated overnight with 10% (w/v) BSA in Tris-buffered saline with 0.1% Tween 20 (TBST) and membranes were then incubated with the anti-TRPC1, anti-TRPC3 or anti-TRPC6 antibody diluted 1:200 in TBST for 2 h, the anti-STIM1 antibody diluted 1:250 in TBST for 2 h or the anti-Orai1 antibody diluted 1:1000 in TBST for 90 min. To detect the primary antibody, blots were incubated with the appropriate horseradish peroxidaseconjugated anti-rabbit IgG or anti-mouse IgG antibody, diluted 1:10,000 in TBST, and exposed to enhanced chemiluminescence reagents for 4 min. Blots were then exposed to photographic films and the optical density was estimated using scanning densitometry. For reprobing, membranes were incubated for 30 min at 50 °C in stripping buffer containing 100 mM 2-mercaptoethanol, 65.5 mM Tris, and 2% SDS, pH 6.7. Membranes were then washed and Western blotting was performed using anti-actin antibody diluted 1:1000 for 2 h as described previously. Statistical analysis Data are expressed as mean ± SEM. Significant difference between individual groups was tested by using the Student's t-test. The level of significance was set at p b 0.05. Results and discussion We have previously reported that Ca2+ entry in platelets is enhanced in DM2 patients compared to controls [10,13]. Here, we have investigated and compared in platelets from DM2 and healthy subjects the expression of different membrane proteins involved in CCE and NCCE mechanisms for Ca2+ influx, including the endoplasmic reticulum Ca2+ sensor, STIM1, and the Ca2+ permeable channel subunits Orai1 and TRPC1, as well as that of TRPC6, which has been shown to participate in CCE as well as NCCE in human platelets, and the TRPC3 protein involved in NCCE [7], by Western blotting. A STIM1immunoreactive band of about 87 kDa was detected in platelet lysates. Interestingly, the amount of STIM1 detected in platelets from DM2 patients was found to be enhanced to 131 ± 12% of controls (Fig. 1A,
Fig. 1. Expression of STIM1, Orai1 and TRPC1 and 6 in platelets from DM2 patients and control subjects. Platelet lysates from control and DM2 donors were subjected to SDS/ 10%-PAGE and Western blotting with anti-STIM1 (A, top panel), anti-Orai1 (B, top panel), anti-TRPC1 (C, top panel) and anti-TRPC6 (D, top panel) antibodies. Membranes were reprobed with anti-actin antibody (bottom panels) for protein loading control. Histograms represent protein expression as percentage of healthy controls and are expressed as average ± SEM of six to eight separate experiments. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel.
H. Zbidi et al. / Blood Cells, Molecules, and Diseases 43 (2009) 211–213
213
agonists, and might overcome the reduced expression of TRPC6, which might explain the altered ionic homeostasis described in insulin resistant conditions [19]. Acknowledgments This work was supported by MCyT-DGI grant BFU2007-60104, Fundesalud (Junta de Extremadura) grant PRI08A003 and Ministerio de Asuntos Exteriores y Cooperación (A/016208/08). J.J.L. was supported by a grant (BFU2004-00165) from the Spanish MEC-DGI. References
Fig. 2. Expression of TRPC3 in platelets from DM2 patients and control subjects. Platelet lysates from control and DM2 donors were subjected to SDS/10%-PAGE and Western blotting with anti-TRPC3 antibody (top panel). Membranes were reprobed with antiactin antibody (bottom panel) for protein loading control. Histograms represent protein expression as percentage of healthy controls and are expressed as average ± SEM of six separate experiments. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel.
other cells [7,15]. We have recently found that the interaction of Orai1/ STIM1 with TRPC6 recruits TRPC6 in the CCE pathway [16]. To test protein loading in samples from DM2 and healthy control donors we performed Western blotting with an anti-actin antibody. As shown in Fig. 1, bottom panels, Western blotting with anti-actin antibody confirmed a similar amount of actin in both lanes (DM2 and controls), which, together with the equal cell concentration used for both experimental groups (2 × 108 cells/mL) support that the different protein detection observed in DM2 and healthy donors by Western blotting is unlikely attributed to different protein loading but to a modification induced by DM2 itself. We have further investigated the expression of other NCCE channel subunits, such as TRPC3, also expressed in human platelets [7]. Here we show that TRPC3 expression was significantly enhanced in platelets from DM2 patients compared to controls (TRPC3 expression in platelets from DM2 patients was 125 ± 8% of that in platelets from healthy donors; Fig. 2, top panel; p b 0.05; n = 6). Western blotting of the same membranes with anti-actin antibody confirms a comparable protein loading in both lanes (Fig. 2, bottom panel). We report for the first time altered expression of a number of proteins involved in CCE and NCCE in platelets from DM2 patients compared with age- and gender-matched healthy donors. Our findings provide an explanation to the altered Ca2+ entry in response to the physiological agonist thrombin or thapsigargin previously reported in platelets from DM2 patients [10]. Thrombin and thapsigargin are able to induce both CCE and NCCE [7,17], the latter (thapsigargin) by stimulating secretion of platelet-activating factors [18]. The increased expression of the CCE complex STIM1/Orai1 and that of the NCCE channel TRPC3 might explain the enhanced Ca2+ entry observed in platelets from DM2 subjects upon stimulation with
[1] M.E. Carr, Diabetes mellitus: a hypercoagulable state, J. Diabetes Complicat. 15 (2001) 44–54. [2] T.J. Rink, S.O. Sage, Calcium signaling in human platelets, Annu. Rev. Physiol. 52 (1990) 431–449. [3] I. Jardin, N. Ben Amor, J.M. Hernandez-Cruz, G.M. Salido, J.A. Rosado, Involvement of SNARE proteins in thrombin-induced platelet aggregation: evidence for the relevance of Ca2+ entry, Arch. Biochem. Biophys. 465 (2007) 16–25. [4] V. Randriamboavonjy, F. Pistrosch, B. Bolck, R.H. Schwinger, M. Dixit, K. Badenhoop, R.A. Cohen, R. Busse, I. Fleming, Platelet sarcoplasmic endoplasmic reticulum Ca2+-ATPase and mu-calpain activity are altered in type 2 diabetes mellitus and restored by rosiglitazone, Circulation 117 (2008) 52–60. [5] J.A. Rosado, F.R. Saavedra, P.C. Redondo, J.M. Hernandez-Cruz, G.M. Salido, J.A. Pariente, Reduced plasma membrane Ca2+-ATPase function in platelets from patients with non-insulin-dependent diabetes mellitus, Haematologica 89 (2004) 1142–1144. [6] I. Jardin, J.J. Lopez, G.M. Salido, J.A. Rosado, Orai1 mediates the interaction between STIM1 and hTRPC1 and regulates the mode of activation of hTRPC1-forming Ca2+ channels, J. Biol. Chem. 283 (2008) 25296–25304. [7] I. Jardin, P.C. Redondo, G.M. Salido, J.A. Rosado, Phosphatidylinositol 4,5bisphosphate enhances store-operated calcium entry through hTRPC6 channel in human platelets, Biochim. Biophys. Acta 1783 (2008) 84–97. [8] A. Braun, D. Varga-Szabo, C. Kleinschnitz, I. Pleines, M. Bender, M. Austinat, M. Bosl, G. Stoll, B. Nieswandt, Orai1 (CRACM1) is the platelet SOC channel and essential for pathological thrombus formation, Blood 113 (2009) 2056–2063. [9] G.M. Salido, S.O. Sage, J.A. Rosado, TRPC channels and store-operated Ca2+ entry, Biochim. Biophys. Acta 1793 (2009) 223–230. [10] F.R. Saavedra, P.C. Redondo, J.M. Hernandez-Cruz, G.M. Salido, J.A. Pariente, J.A. Rosado, Store-operated Ca2+ entry and tyrosine kinase pp60(src) hyperactivity are modulated by hyperglycemia in platelets from patients with non insulindependent diabetes mellitus, Arch. Biochem. Biophys. 432 (2004) 261–268. [11] P.C. Redondo, M.T. Harper, J.A. Rosado, S.O. Sage, A role for cofilin in the activation of store-operated calcium entry by de novo conformational coupling in human platelets, Blood 107 (2006) 973–979. [12] P.C. Redondo, G.M. Salido, J.A. Pariente, J.A. Rosado, Dual effect of hydrogen peroxide on store-mediated calcium entry in human platelets, Biochem. Pharmacol. 67 (2004) 1065–1076. [13] A. Bouaziz, S. Salido, P.J. Linares-Palomino, A. Sanchez, J. Altarejos, A. Bartegi, G.M. Salido, J.A. Rosado, Cinnamtannin B-1 from bay wood reduces abnormal intracellular Ca2+ homeostasis and platelet hyperaggregability in type 2 diabetes mellitus patients, Arch. Biochem. Biophys. 457 (2007) 235–242. [14] G.M. Salido, S.O. Sage, J.A. Rosado, Biochemical and functional properties of the store-operated Ca2+ channels, Cell. Signal. 21 (2009) 457–461. [15] T. Hofmann, A.G. Obukhov, M. Schaefer, C. Harteneck, T. Gudermann, G. Schultz, Direct activation of human TRPC6 and TRPC3 channels by diacylglycerol, Nature 397 (1999) 259–263. [16] I. Jardin, L.J. Gomez, G.M. Salido, J.A. Rosado, Dynamic interaction of hTRPC6 with the Orai1/STIM1 complex or hTRPC3 mediates its role in capacitative or noncapacitative Ca2+ entry pathways, Biochem J (2009) doi:10.1042/BJ20082179. [17] J.A. Rosado, S.O. Sage, Protein kinase C activates non-capacitative calcium entry in human platelets, J. Physiol. 529 (Pt. 1) (2000) 159–169. [18] A.G. Harper, S.L. Brownlow, S.O. Sage, A role for TRPV1 in agonist-evoked activation of human platelets, J. Thromb. Haemost. 7 (2009) 330–338. [19] L.M. Resnick, Ionic basis of hypertension, insulin resistance, vascular disease, and related disorders. The mechanism of “syndrome X”, Am. J. Hypertens. 6 (1993) 123S–134S.